PROJECT SUMMARY Our laboratory investigates the functional organization of mammalian membranes to generate mechanistic insight into the connections between lipid composition, membrane structure, and cell physiology. Membranes host a major fraction of all cellular bioactivity, orchestrating myriad simultaneous, parallel tasks. This functionality is enabled by the remarkable complexity and diversity of mammalian lipidomes, which give rise to a unique combination of biophysical phenotypes, including membrane fluidity, tension, curvature, and lateral compartmentalization. We aim for a predictive understanding of how lipidomic features determine membrane properties, and how these in turn regulate cell functions. Progress towards this goal has been enabled by recent methodological advances in high-throughput lipidomics, biophysical analysis of plasma membranes, and quantitative high-resolution spectral microscopy. Using these, we have made significant advances in understanding several interrelated aspects of lateral and transverse organization of mammalian membranes. We have defined broad structural features responsible for protein association with ordered membrane domains and how such domains are involved in membrane traffic. Despite these insights, there remains remarkably little known about which proteins associate with membrane domains and why. Our experimental and computational framework allows us to address key outstanding questions, including: what are the structural codes for protein affinity for ordered domains, and how does protein association with membrane domains facilitate their localization and function? In parallel, we have characterized the remarkable plasticity of mammalian membranes (particularly their susceptibility to dietary fatty acids) and characterized the effects of external inputs on membrane properties and cell function. However, how cells maintain membrane homeostasis in response to continuous challenge from dietary and other external inputs is not well understood. Our observations suggest that interference with such homeostatic mechanisms may provide a novel therapeutic strategy for treatment of cardiovascular disease or cancer. Finally, our most recent work has focused on the asymmetric distribution of lipids between the two leaflets of the plasma membrane bilayer. Although such compositional asymmetry appears to be a universal design principle for living membranes, the selective advantages that asymmetry confers are not known. We are defining the compositional, biophysical, and functional asymmetry of the plasma membrane in mammalian cells to answer major open questions about the biophysical consequences of membrane asymmetry and how physical properties are coupled across asymmetric leaflets. Functionally, intriguing recent discoveries reveal that transient loss of membrane asymmetry is widespread during immune activation, and is necessary for optimal response; however, the mechanisms underlying these effects of transient lipid scrambling are unknown. Addressing these questions will advance our understanding of the functional role of membrane organization across the tree of life.